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The promoter of the long variant of collagen XVIII, the precursor of endostatin, contains liver-specific regulatory elements
The Promoter of the Long Variant of Collagen XVIII, the Precursor of Endostatin, Contains Liver-Specific Regulatory Elements JOCELYNE LIE´ TARD,1,2 NATHALIE THE´ RET,1 MARKO REHN,2 ORLANDO MUSSO,1 DELPHINE DARGE` RE,1 TAINA PIHLAJANIEMI,2 AND BRUNO CLE´ MENT1
The endostatin precursor collagen XVIII is expressed at high levels in human livers, the main source being hepatocytes. We have studied the regulatory elements in the promoter 2 of the Col18a1 gene that directs the transcription of the NC1-517 variant of collagen ␣1(XVIII), which is the main form expressed in the liver. The 5ⴕ-flanking region of Col18a1 gene was cloned, and a series of 5ⴕ-deletions from ⴚ3286 bp to ⴙ285 bp were linked to the luciferase reporter gene. Transfection experiments in HepG2 cells allowed to identify a silencer-like element containing putative HNF1 and HNF3 sites and activator elements containing stretches of GC-rich sequences. Another putative HNF3 site in close apposition to a NF1/CTF site was localized upstream of the silencer-like element. Cotransfection experiments showed that the Col18a1 promoter 2 was transactivated by Sp1 and HNF3␣. Gel-shift analyses showed that HNF3, NF1/CTF, and Sp1-like sites specifically recognized nuclear factors. Super-shift experiments indicated that HNF3 was the major form of HNF3 interacting with the HNF3/NF1 site. The well-differentiated hepatoma cell line mhATFS315 transfected with a truncated form of HNF3, which competitively blocks HNF3 transactivating activity, expressed the Col18a1gene at a very low level. Taken together, these data strongly suggest that Col18a1 is a liver-specific gene. Furthermore, gel-shift analyses performed with nuclear factors prepared from well-differentiated hepatocellular carcinomas showed increased HNF3/NF1 binding activity compared with normal livers. Consequently, the precursor of endostatin might be differently expressed according to the
Abbreviations: HNF, hepatocyte nuclear factor; CMV, cytomegalovirus; cDNA, complementary DNA; CAT, chloramphenicol acetyl transferase; TLC, thin-layer chromatography; EDTA, ethylenediaminetetraacetic acid; EGTA, ethyleneglycoltetraacetic acid; TBE, tris/borated/EDTA; mRNA, messenger RNA; TTR, transthyretin. From the 1Detoxication and Tissue Repair Unit, INSERM U-456, Universite´ de Rennes I, France; and 2Collagen Research Unit, Biocenter and Department of Medical Biochemistry, University of Oulu, Oulu, Finland. Received May 18, 2000; accepted September 7, 2000. Supported by INSERM, DRRC Bretagne (CHU Pontchaillou, Rennes), Association pour la Recherche contre le Cancer (France), the Health Sciences Council of the Academy of Finland, the Sigrid Juselius Foundation, and FibroGen Inc. (South San Francisco, CA). Sequence data from this article has been submitted to the GenBank/EMBL Data libraries with accession number No 34607. Drs. Lie´tard and The´ret contributed equally to this work. Address reprint requests to: Nathalie The´ret, Ph.D., INSERM U-456, Universite´ de Rennes I, 2 ave. Le´on Bernard, 35043 Rennes Cedex, France. E-mail: [email protected]; fax: (33) 2-99-33-62-42. Copyright © 2000 by the American Association for the Study of Liver Diseases. 0270-9139/00/3206-0024$3.00/0 doi:10.1053/jhep.2000.20066
differentiated and/or transformed state of hepatocytes. (HEPATOLOGY 2000;32:1377-1385.) Angiogenesis is involved in most physiological and pathological processes and is essential for tumor growth and metastasis.1,2 It depends on a balance between stimulatory factors, e.g., vascular endothelial growth factor and fibroblast growth factors, and inhibitory factors, e.g., thrombospondin-1, SPARC, fibronectin fragments, angiostatin, and endostatin.3 We have recently proposed that the endogenous regulation of angiogenesis is a newly identified liver function,4 as endostatin, angiostatin, the cleaved form of antithrombin,5 the kringle-2 domain of prothrombin,6 and the domain 5 of kininogen7 are cryptic angiogenesis inhibitors within parent plasma proteins involved in the clotting and fibrinolytic cascades and are secreted by hepatocytes. Endostatin8,9 is proteolytically derived from the C-terminal domain of the nonfibrillar collagen XVIII10-12 which is associated with basement membranes13 and is secreted as a plasma protein.10 Collagen XVIII is expressed at high levels by human adult hepatocytes, and its expression is increased early in the onset of liver fibrosis.14,15 Human and mouse collagen ␣1(XVIII) genes are located on chromosome 21q22.3 and 10, respectively.16 The complete organization of the 102 kb mouse Col18a1 gene was recently reported.17 Mouse collagen XVIII transcripts encode polypeptides that differ in size with respect to the 3 variant N-terminal noncollagenous domains that are 301 (NC1-301), 517 (NC1517), or 764 (NC1-764) residues in length.18,19 The 3 variant NC1 domains result from the use of 2 alternative promoters, separated by a distance of 50 kb.17 The upstream promoter, promoter 1, directs the synthesis of the NC1-301 domain. The downstream promoter, promoter 2, directs that of the NC1517 and NC1-764 domains, with the latter 2 variants differing with respect to alternative splicing.17 Human hepatocytes express the NC1-517 variant of collagen ␣1(XVIII),13,14 which is almost exclusively detected in adult and fetal livers at high levels, suggesting that its expression is liver-specific in humans.20 However, neither the 5⬘-flanking regions of the Col18a1 gene nor the factors that govern its striking hepatocyte-specific expression have been characterized so far. In the present study, we have identified potential regulatory elements implicated in the regulation of the transcription of Col18a1 gene in HepG2 cells after cloning the 5⬘-flanking region of promoter 2, which governs the expression of the NC1-517 variant. We show that, as in other genes specifically expressed in the liver, both Sp1 (ubiquitous) and HNF3/NF1 (liver-specific) regulatory elements are involved in the combinatorial control of the expression of collagen XVIII and that
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HNF3/NF1 binding activity is increased in well-differentiated human hepatocellular carcinomas. MATERIALS AND METHODS Sequence Analysis of the 5ⴕ-Flanking Region of the Downstream Promoter From the Mouse Collagen ␣1(XVIII) Gene A 5.3-kb HindIII fragment of the P1 clone P1-212417 was digested with NaeI, and the resulting 3.6-kb HindIII/NaeI fragment covering the promoter 2 sequences of the Col18a1 was subcloned into the vector pSP72 (Promega, Madison, WI). Sequencing from both strands was performed with ABI PRISM 377 Automated Sequencer (Perkin-Elmer, Applied Biosystems, France) and consensus sites for the binding of transcription factors were searched with Mat Inspector Prog. (Transfac database). Plasmid Constructs Several recombinant plasmids containing the 5⬘-flanking sequences of the gene coding for the 2 longest variants of collagen XVIII fused to the structural part of the gene encoding luciferase were constructed. The vector pSP72 has been first modified as follows: pSP72 was linearized with XhoI/EcoRI, a linker containing restriction sites was attached (XhoI-SalI-MluI-HindIII-NaeI-EcoRI), digested with HindIII/NaeI and then the 3.6-kb HindIII/NaeI fragment mentioned earlier was ligated. The plasmid was then digested with MluI and protected with dNTPs, digested with HindIII and the double-stranded deletions were prepared using the “double-stranded nested deletion kit” (Pharmacia) according to the manufacturer. Segment deletions were selected and subcloned into the SalI/SmaI site of pXP2 vector.21 The 3.6-kb fragment in pSP72 and the different deletion constructs used in this study, i.e., del 7 (⫺3286 bp to ⫹285 bp), del 9 (⫺2835 bp to ⫹285 bp), del 10 (⫺1843 bp to ⫹285 bp), del 1.3 (⫺1329 bp to ⫹285 bp), del 2.1 (⫺935 bp to ⫹285 bp), and del 12 (⫺492 bp to ⫹285 bp), were sequenced from both strands using ABI PRISM 377 Automated Sequencer (Perkin-Elmer). The human expression vector for Sp1 under the control of the CMV promoter, pEVR2/CMV/Sp1 and pCMV-HNF3␣ were generous gifts of Dr. Suske (Institut fu¨r Molekularbiologie und Tumorforschung, Germany) and Dr. Costa (University of Illinois at Chicago), respectively. All plasmids used for transfection were purified by cesium chloride centrifugation steps. Cell Culture Human hepatoblastoma (HepG2) cells were routinely maintained in Williams’s medium (Eurobio, France) supplemented with 10% fetal calf serum and 1% streptomycin at 37°C. Only confluent cells were used in the study. The well-differentiated mhAT3F hepatoma cell line was derived from the tumorous liver of a transgenic mouse expressing simian virus 40 early genes under the control of the liverspecific antithrombin III promoter.22 The mhAT3F S315 consisted of stably transfected mhAT3F with a truncated HNF3 protein expression vector resulting in competition with the endogenous HNF3 proteins and subsequently in inhibition of liver-specific genes.23 Both the wild type, mhAT3F, and the transfected mhATFS315 hepatoma cell lines were obtained from Dr. B. Antoine (INSERM U129, Institut Cochin, Paris) and cultured as previously described.23 Liver Samples Liver tissue samples were obtained from patients at the Department of Surgery, Hoˆpital Pontchaillou, Rennes, France, between November 1994 and July 1997. Tumor samples from 6 patients with hepatocellular carcinoma were analyzed. Normal samples (n ⫽ 3) were from liver donors who had been excluded from the transplantation protocol because of extra-abdominal infection unrelated to the cause of death. The absence of significant histological lesions was verified before inclusion of these samples as controls. All specimens were routinely processed for histology, i.e., hematoxylin-eosin-saffran and Sirius red staining. Access to this biopsy material was in
HEPATOLOGY December 2000 TABLE 1. Oligonucleotides Used in This Study Sp1C18 (⫺818 bp) Sp1cons HNF3C18 (⫺2788 bp) HNF3alb HNF3TTr NF1C18 (⫺2778 bp) HNF3NF1C18 (⫺2778 bp)
5⬘-GGGGTCAGGGCGGCTATGTG-3⬘ 5⬘-ATTCGATCGGGGCGGGGCGAGC-3⬘ 5⬘-TCCAGCTGTTTGCAGCTGGA-3⬘ 5⬘-CCGAACGTGTTTGCCTTGGCC-3⬘ 5⬘-GTTGACTAAGTCAATAATCAGAATCAG 5⬘-AGCTGGACTCTGAGCCAAA-3⬘ 5⬘-TCCAGCTGTTTGCAGCTGGACTCTGAGCCAAA-3⬘
NOTE. Binding sites containing sequences from Col18␣1 promoters (Sp1C18, HNF3C18, NF1C18, and HNF3NF1C18) and from albumin (HNF3alb) and transthyretin (HNF3TTr) gene promoters are underlined. Sp1 consensus is commercially available from Santa Cruz Biotechnology.
agreement with French laws and satisfied the requirements of the Ethics Committee of the institution. Transient Transfections and Luciferase Assays HepG2 cells were transfected with plasmid DNA by electroporation using a Gene Pulser apparatus (Eurogentec, Seraing, Belgium). Luciferase plasmids (30 g) were cotransfected with 10 g of pRSVCAT plasmid to normalize for transfection efficiencies. For cotransfection experiments, 30 g of luciferase plasmids were cotransfected with either 10 g of pEVR2/CMV/Sp1 plasmid, 10 g of pCMV/ HNF3␣ plasmid or 10 g of the control expression vectors (without the respective cDNA). Cells were harvested 48 hours after transfection, and luciferase activity was determined from cell extracts using the luciferase assay system (Promega, Madison, WI). CAT assays were carried out by the method of Gorman et al.24 Radioactive spots on TLC plates were cut out and quantitated by scintillation counting. Nuclear Protein Extracts and Gel Shift Assays Nuclear extracts from HepG2 cells and from liver samples were prepared, and gel shift assays were performed as described by Cereghini et al.25 Binding reactions were carried out in a 15 L volume containing 1 mmol/L sodium phosphate, pH 7.5, 0.1 mmol/L ethylenediaminetetraacetic acid (EDTA), 0.5 mmol/L ethyleneglycoltetraacetic acid (EGTA), 0.5 mmol/L dithiothreitol, 10% (vol/vol) glycerol, 2 g poly (dI-dC), 1 mmol/L MgCl2, 10 mmol/L spermidine, and 20,000 cpm of P-labeled 5⬘-end double-stranded oligonucleotide. Oligonucleotides from the Col18a1 gene promoter 2, and from both HNF3 albumin26 and HNF3 transthyretin27 gene promoters are presented in Table 1. The Sp1 consensus oligonucleotide was purchased from Santa Cruz Biotechnology and was termed Sp1cons. Five micrograms of nuclear proteins were added to the reaction mixture and incubated at room temperature for 20 minutes. The DNA-protein complexes were resolved on 6% acrylamide gels in 0.5 ⫻ TBE (45 mmol/L Tris-borate, 1.25 mmol/L EDTA) at 25 mA. Then, gels were fixed, dried, and subjected to autoradiography. For supershift experiments, 5 g of nuclear proteins and radiolabeled oligonucleotides were incubated at room temperature for 20 minutes in 10 mmol/L Tris (pH 7.5) buffer with 50 mmol/L NaCl, 1 mmol/L dithiothreitol, 1 mmol/L EDTA, 5% glycerol and 1 g poly (dI-dC). One microliter of specific anti HNF3␣, HNF3, or HNF3␥ antibody (kindly provided by Dr. G. Schutz, German Cancer Research Center, Heidelberg, Germany) and 1 or 5 L of specific antiNF1/CTF antibodies (kindly provided by Dr. M. Raymondjean, INSERM U129, Institut Cochin, Paris) were then added and incubated at room temperature for 45 minutes. Northern and Dot Blot Analyses Total RNA from cells were prepared by using the guanidium thiocyanate/phenol-chloroforme method.28 Northern and dot blots were
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FIG. 1. Sequence of the 5⬘-flanking region of the promoter 2 from the mouse Col18a1 gene coding the polypeptide forms NC1-517 and NC1-764. The nucleotides are numbered from the extreme upstream major transcription initiation site. Horizontal bars indicate the locations of consensus sequences for binding sites of certain transcription factors. The CTC-rich sequences are in italics.
performed as previously described.29 cDNA Collagen XVIII probe was a 458 bp cDNA, which recognized the long variant of the mouse ␣1(XVIII) chains.19 A 25-mer oligoprobe corresponding to 18S ribosomal RNA was used30 for standardization of the signals.
latter being in close apposition with a NF1/CTF site at ⫺2778 bp.
RESULTS
Potential regulatory elements responsible for governing transcription of the Col18a1 gene were identified in HepG2 cells. A series of 5⬘ deletions from ⫺3286 bp to ⫹285 bp was constructed from the mouse promoter and linked to the luciferase reporter gene. Transfection experiments were carried out in 3 independent experiments (Fig. 2). The del 7 plasmid gave a transcriptional activity that was arbitrarily chosen as 100%. The del 9 plasmid showed a slightly lower activity. Truncation of the upstream region from ⫺2835 bp to ⫺1329 bp resulted in increased luciferase activities after transfection of del 10 and del 1.3, respectively. Truncation of the putative Sp1-binding site localized at ⫺1068 bp, and transfection of del 2.1 construct resulted in a weaker luciferase activity compared with the del 1.3 plasmid. Transfection of the shortest construct, del 12 from ⫺492 bp to ⫹285 bp gave a marked decrease in luciferase activity. This suggests that a silencerlike element(s) is present between ⫺2835 bp and ⫺1329 bp,
Sequence Analysis of the 5ⴕ-Flanking Region of the Downstream Promoter From the Mouse Collagen ␣1(XVIII) Gene
The sequence of 3.4 kb upstream of the transcription start site of the promoter 2 was determined (Fig. 1). The gene lacked a TATA box upstream of the transcription start site, but contained 2 CCAAT boxes at ⫺65 bp and ⫺148 bp, with the latter one in reverse orientation. The gene also contained several CTC-rich sequences, including a stretch between ⫺2058 bp and ⫺2170 bp and 3 boxes localized at ⫺1026 bp, ⫺1855 bp, and ⫺3049 bp, respectively. Potential binding sites for transcription factors were identified including several Sp1 binding sites, 1 AP-1 binding site, and 2 potential binding sites for c-Krox factor.31 In addition, 1 HNF1 potential binding site was found at ⫺1918 bp, and 2 HNF3 binding sites were localized at ⫺1746 bp and ⫺2788 bp, respectively, the
Deletion Analysis of the Col18a1 Promoter
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FIG. 2. Transfection analysis of the Col18a1 promoter 2 in HepG2 cell line. A series of deletion constructs and their corresponding luciferase activities are represented on the left. Relative luciferase activities are given as percentages of del 7 plasmid activity. The different deletion plasmids were cotransfected with the expression vectors coding HNF3␣ and Sp1. The transactivation of the different fragments was evaluated by measuring the ratio of the relative luciferase activity in presence of the expression vector versus that obtained in presence of the control vector (right panel).
and a transcriptional activator element between ⫺1329 bp and ⫺492 bp, which contains several GC-rich elements and an AP-1 site. Cotransfections With Sp1 and HNF3␣ Expression Vectors
To investigate whether Sp1 and HNF3␣ have a potential role in the regulation of the Col18a1 gene, the different luciferase constructs were cotransfected with corresponding expression vectors. Results for each deletion construct are expressed as a ratio of the relative luciferase activity obtained with the expression vector to that obtained with the control (Fig. 2). Cotransfection with the Sp1 expression vector markedly increased the activity of all the constructs containing GC-rich elements. The 2 longest constructs containing the upstream HNF3 site, i.e., del 7 and particularly del 9, were
FIG. 3. (A) Gel shift assays. Nuclear extracts were prepared from HepG2 cells and incubated with specific oligonucleotides. (A, left panel) 32P-labeled oligonucleotide Sp1C18 (lanes 2 to 4): Incubation steps were performed without (-) or with 100fold excess of Sp1C18 (lane 3) and Sp1cons (lane 4) unlabeled oligonucleotides. Lane 1: control without nuclear extracts. Arrows on the left indicate specific complexes. NS, non-specific complex. (A, right panel) 32P-labeled oligonucleotides SP1consensus (lanes 1 to 3). Incubation steps were performed with 100 (lane 2) or 50 fold-excess (lane 3) of Sp1C18 oligonucleotides. (B) Northern blot analyses of endogenous collagen ␣1(XVIII) mRNA expression in HepG2 cells transfected with either pEVR2-CMV-Sp1 (lane 2) or pEVR2-CMV (lane 3). Controls were electroporated cells in the absence of plasmid (lane 1). The ratio collagen XVIII/18S were evaluated by densitometry scanning.
induced by HNF3␣, whereas the activity of other plasmids was not changed. These results suggest that the Col18a1 gene is transactivated with Sp1 and that HNF3␣ can be implicated in the regulation of the Col18a1 gene through the upstream HNF3 site located at ⫺2788 bp. Sp1 Binding Activity in HepG2 Cells
The interactions of endogenous Sp1 factor(s) with the Col18a1 promoter was investigated by gel-shift assays with HepG2 nuclear factors. Oligonucleotide-termed Sp1C18 (Table 1) correspond to sequence in the promoter region containing putative Sp1-binding site localized at ⫺818 bp. Two complexes (arrows in Fig. 3A, left panel, lane 2) were resolved by gel-shift assay with 32P-labeled Sp1C18 probe. These complexes were competed by a 100-fold molar excess of unlabeled
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competitor Sp1C18 (lane 3) or Sp1consensus (lane 4). Complexes of similar sizes were found with SP1cons oligonucleotides (Fig. 3A, right panel, lane 1) and were competed by a 100-fold molar excess of unlabeled competitor Sp1C18 (lane2), the 2 Sp1-specific complexes being still detectable in the presence of 20-fold molar excess of competitor (Fig. 3A, right panel, lane 3). These results suggest that the GC-rich sequence at ⫺818 bp interacts with Sp1-related factor(s). To assess whether Sp1 modulate collagen ␣1(XVIII) mRNA expression, HepG2 cells were transfected with a Sp1 expression vector (Fig. 3B). Control was electroporated cells in the absence of plasmid, and assays were carried out with either pEVR2-CMV-Sp1 or pEVR2-CMV construct. The steady-state collagen ␣1(XVIII) mRNA levels were 2.3-fold and 3.6-fold increased when cells were transfected with Sp1 expression vector (lane 2) compared with control (lane 1) and Sp1-less expression vector (lane 3), respectively. This suggests that Sp1 acts as a positive regulatory element for the transcription of Col18a1 gene. HNF3 and NF1/CTF Binding Activity
The recognition sequence for the NF1/CTF family of transcription factors, TGGAN7CCA is located 3 bp downstream of the HNF3 putative site localized at ⫺2788 bp. Because it has been shown in other systems that interactions between HNF3 and NF1 in close apposition occurs in liver-specific genes,32 gel-shift experiments were performed with different oligonucleotides (Table 1). The first corresponds to the HNF3 site at ⫺2788 bp in the Col18a1 gene (oligonucleotide HNF3C18), the second corresponds to the 2 putative HNF3 and NF1/CTF sites in close apposition (oligonucleotide HNF3NF1C18). The third corresponds to the NF1/CTF site (oligonucleotide NF1C18). HNF3C18. Two major complexes, termed H1 and H2, were detected by gel-shift assay with 32P-labeled HNF3C18 oligonucleotide (Fig. 4A, lanes 1 and 11). H1 complexes were competed by a 60-fold excess of unlabeled HNF3C18 oligonucleotides (lanes 2 and 14) whereas competition with H2 complexes required higher concentration of oligonucleotides (lanes 3 and 4). Binding of HNF3C18 was only partially competed by oligonucleotides corresponding to the binding sites in the mouse transthyretin (HNF3TTR) and albumin (HNF3alb) genes (lanes 5 to 10). The complex H1 was completely competed by an excess of unlabeled HNF3NF1C18 (Fig 4A, lane 12), and slightly competed by an excess of unlabeled NF1C18 (lane 13), the second complex H2 being partially competed by HNF3NF1C18 and NF1C18 oligonucleotides (lanes 12 and 13, respectively). NF1C18. Two complexes were detected by gel-shift assay with 32P-labeled NF1C18 oligonucleotide (Fig. 4B, lanes 1 and 4). These complexes had the same shift than complexes H1 and H2, H1 being totally and H2 partially competed by an excess of both cold HNF3NF1C18 (lane 5) and NF1 (lane 6) oligonucleotides. An excess of cold HNF3C18 oligonucleotide did not show competition (lane 7). HNF3NF1C18. Three complexes were detected by gel-shift assay with 32P-labeled HNF3NF1C18 oligonucleotide (Fig. 4B, lanes 3 and 8), including H1 and H2 and an additional complex, X. The complex X was completely competed by an excess of unlabeled HNF3NF1C18 (Fig. 4B, lane 9), NF1C18 (lane 10) and HNF3C18 oligonucleotides (lane 11). Complex H1 was totally competed by an excess of cold HNF3NF1C18 (lane 9), being only partially reduced by an excess of cold
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NF1C18 (lane 10) and not competed by an excess of cold HNF3C18 oligonucleotides (lane 11). The signal intensity of complex H2 was reduced by HNF3NF1C18 and NF1C18 oligonucleotides (lane 9 and 10, respectively) but not affected by HNF3C18 oligonucleotides (lane 11). Taken together, the data indicate that several nuclear factors are involved in the formation of the 2 complexes H1 and H2, which interact with HNF3- and NF1-like regulatory elements between ⫺2794 bp and ⫺2751 bp. Complex X probably corresponds to a supra-arrangement of nuclear factors, which cooperate for interacting with the HNF3/NF1 site. Importantly, the data suggest that both HNF3- and NF1-binding sites overlaps, as previously shown in other liver-specific genes. The affinity of complex interactions with HNF3/NF1like motif appears modulated by NF1-related factors. Involvement of HNF3 in the Regulation of Col18a1 Expression
To further characterize the complexes H1, H2, and X, nuclear extracts from HepG2 were incubated with radiolabeled HNF3C18 or HNF3NF1C18 oligonucleotides in the presence of either specific anti-HNF3␣, HNF3, HNF3␥, or NF1 antibodies (Fig. 5). The lower intensity of complexes H2 in supershift assay conditions compared with those in shift assays may be related to the differences in stringency between the 2 reaction mixtures, which greatly affect stability of complexes H2. When anti-HNF3 antibodies were added to the reaction mixture with radiolabeled HNF3C18 (Fig. 5A), a specific decrease in the intensity of the H1 complex was observed, the intensity of complex H2 being enhanced (Fig. 5A, lane 3). No significant change was found with either HNF3␣ or HNF3␥ antibodies (Fig. 5A, lanes 2 and 4). A similar pattern was observed when HNF3TTR and HNF3alb oligonucleotides were used as probes (Fig. 5A). With HNF3NF1C18 and NF1C18 oligonucleotides, similar shifts of H1 and H2 complexes were observed in the presence of HNF3 antibodies (Fig. 5B, lanes 3 and 9). In addition, the complex X was strongly reduced. Addition of NF1 antibodies prevented the formation of complex X with HNF3NF1C18 oligonucleotides (Fig. 5B, lanes 5 and 6); a slight decrease in the intensity of complex H1 was found with NF1C18 oligonucleotides (Fig. 5B, lane 12), being more obvious with HNF3C18 oligonucleotides (Fig. 5B, lane 18). Despite a low intensity of H2 complexes in supershift assay conditions, NF1 antibodies appeared to affect the formation of H2 complexes (Fig. 5B, lane 18). The expression of collagen ␣1 (XVIII) was investigated in the mouse hepatoma cell line mhATF. This cell line was stably transfected with a truncated form of HNF3, which competed with endogenous HNF3 for binding DNA sites, thus resulting in a decrease in hepatospecific gene expression.23 As shown in Fig. 6, the steady-state collagen ␣1(XVIII) mRNA was significantly lower in transfected versus nontransfected cells, similarly to albumin mRNA levels. Taken together, the data indicate that HNF3 is the major form of HNF3 involved in the formation of the protein complexes that regulate Col18a1 promoter 2 in HepG2 cells and that this regulatory factor is necessary for a stable expression of Col18a1 gene. In addition, HNF3␣ is also able to recognize the regulatory elements as shown by the induction of del 7 and del 9 transcription by HNF3␣ expression vector. Thus, Col18a1 gene appears to be regulated by nuclear factors that modulate liver-specific gene expression.
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Changes in HNF3/NF1 Binding Activity in Hepatocellular Carcinomas
The significance of HNF3/NF1 binding activity was further studied in human liver biopsies by gel shift assays. Nuclear extracts from 3 control livers and 6 hepatocellular carcinomas were incubated with radiolabeled oligonucleotides (Fig. 7). Three complexes comigrating with those observed in HepG2 cells nuclear extracts were found: H1, H2, and X, the intensity of the latter being very low in control liver and weaker in tumor samples compared with HepG2 cells. A striking difference between tumor and control livers was an increase in binding activities in hepatocellular carcinoma samples, thus suggesting the involvement of these complexes in regulating collagen ␣1 (XVIII) expression in cancer liver. DISCUSSION
We have analyzed the molecular mechanisms that govern the expression of the ␣1 chain of collagen XVIII, by cloning the promoter 2 of Col18a1 gene that encodes the NC1-517 domain, i.e., the major collagen XVIII variant in the liver. We found that the 5⬘-flanking region of the downstream promoter 2 of Col18a1 gene contains 2 CCAAT boxes and several putative Sp1-binding sites, which are also found in the regulatory regions of both COL1A1 and COL1A2 that encode collagen ␣1(I) and ␣2(I) transcripts, respectively33-36 and in collagen IV genes.37,38 Different putative Sp1-binding sites were identified, and one of them located at ⫺818 bp is able to bind an Sp1-related factor(s). An Sp1 expression vector was able to transactivate the different constructs that contain GC-rich boxes and to act as a positive regulator on basal collagen ␣1 (XVIII) mRNA expression in the human hepatoma cell line, HepG2. In addition, several CTC boxes, another element found in the regulatory regions of various collagen genes, including COL1A1,34,35 COL1A2,33 COL4A1,37,38 and LAMC1 that encodes the laminin ␥1 chain39 were also present. Two potential binding sites for c-Krox with the consensus sequence GGGAGGG31 were also identified. It has been shown that c-Krox can bind to several sites in the promoter of both mouse collagen I genes,31,40 but whether the 2 sites in the Col18a1 gene are involved in its regulation needs further investigation. Besides these ubiquitous sites found in the regulatory regions of various collagen and housekeeping genes, potential binding sites for the liver-enriched transcription factors HNF1 and HNF3 were identified in the promoter 2 of Col18a1. Genes that are preferentially expressed in the liver, e.g., albumin, ␣-antitrypsin, aldolase B41 are under the combinatorial control of several families of liver-enriched transcription factors, including the leucine zipper C/EBP 4™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™ Fig. 4. Analysis of the 2 putative HNF3 and NF1/CTF binding sites in close apposition in the Col18a1 promoter 2. Nuclear extracts were prepared from HepG2 cells and incubated with 32P-labeled oligonucleotides. (A) Competition of HNF3C18 binding site by 60, 300, and 1,000 molar excess of unlabeled oligonucleotides. HNF3C18 lanes 2, 3, and 4; HNF3TTR lanes 5, 6, and 7; and HNF3alb, lanes 8, 9, and 10, respectively; by an excess of cold competitor HNF3NF1C18 (lane 12), NF1C18 (lane 13), HNF3C18 (lane 14) oligonucleotides. (B) Comparison of complex formation by gel shift assays with NF1C18 (lane 1), HNF3C18 (lanes 2), HNF3NF1C18 (lanes 3). Complexes H1, H2, and X are indicated on the left. Competition of NF1C18 and HNF3NF1C18 binding sites by an excess of cold competitors HNF3NF1C18 (lanes 5 and 9), NF1C18 (lanes 6 and 10), and HNF3C18 (lanes 7 and 11) oligonucleotides.
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FIG. 5. Gel mobility supershift assays with HNF3 and NF1 antibodies. Nuclear extracts were prepared from HepG2 cells and incubated with 32P-labeled oligonucleotides. (A) HNF3C18 (lanes 1 to 4), HNF3TTRC18 (lanes 5 to 8), and HNF3alb (lanes 9 to 12). Incubation steps were performed without (lanes 1, 5, and 9) or with HNF3␣ (lanes 2, 6, and 10), HNF3 (lanes 3, 7, and 11), HNF3␥ (lanes 4, 8, and 12) antibodies. (B) HNF3C18 (lanes 1 to 6), NF1C18 (lanes 7 to 12), and HNFNF1C18 (lanes 13 to 18). Incubation steps were performed without (lanes 1, 7, and 13) or with HNF3␣ (lanes 2, 8, and 14), HNF3 (lanes 3, 9, and 15), HNF3␥ (lanes 4, 10, and 16), NF1 (1 L) (lanes 5, 11, and 17), NF1 (5 L) (lanes 6, 12, and 18) antibodies. Specific complexes H1, H2, and X are indicated on the left. The lower staining intensity of complex H2 compared with that in Fig. 4 is related to the differences in buffer composition.
(CCAAT/enhancer binding protein), HNF3, HNF1, and HNF4 families.41,42 These families are characterized by structurally related, DNA-binding domains that control the formation of the hepatic endoderm during development and the phenotype of adult hepatocytes.43 The architecture of these promoter regions consists of clusters of liver-specific and ubiquitous transcription factors within short regions, which allow the regulation of transcription through the synergy of both classes of transactivators. We found that HNF3 in combination with NF1/CTF are important regulator elements of the murine Col18a1 promoter 2 activity. Importantly, a putative HNF3-NF1/CTF site is also present in the promoter 2 of the human COL18A1 gene, at about the same distance upstream of the transcription start site mouse (Elamaa H., Lie´tard J. and Pihlajaniemi T., unpublished data, 1999). The HNF3 family is composed of 3 isoforms, namely HNF3␣, HNF3, and HNF3␥, which are encoded by 3 distinct genes that are believed to participate in the liver-specific expression
FIG. 6. Dot blot analyses of endogenous expression of collagen XVIII in the mhATF (䊐) and in the deficient HNF3 mhATFS315 (■) cell lines. Albumin mRNA was used as hepatospecific gene control. Values from densitometric scanning were expressed as mean ⫾ SD of Collagen ␣1(XVIII)/18S and albumin/18S ratios (n ⫽ 5). Student’s t test was used to test the significance of the difference between means: **P ⬍ .01; ***P ⬍ .001.
of a number of genes.27,44,45 This transcription factor family is characterized by a highly conserved DNA-binding domain.46 HNF3␣, , and ␥ recognize 2 apparently unrelated DNA sequence motives, 1 is defined by the high-affinity site of the transthyretin (TTR) promoter,27 the other being the TGT3 motif.32,47,48 In the present study, we show that cotransfection with the HNF3␣ expression vector resulted in an induced activity of the 2 constructs containing the upstream HNF3 site. In apparent contradiction, a silencer-like region was identified between ⫺2835 bp and ⫺1329 bp, as indicated by transfection experiments of deleted plasmids. These observations suggest that the silencer element(s) is likely located downstream of the HNF3 site at ⫺2788 bp. In support of this, the HNF3 expression vector had no effect on the del 10 construct, which contains the second HNF3 site, located at ⫺1746 bp. Thus, the upstream HNF3 site, i.e., at ⫺2788 bp might positively regulate the activity of the promoter. Interestingly, super-shift experiments indicated that HNF3 was the major HNF3 polypeptide in HepG2 to bind the HNF3 sequence in this region. This does not contradict data from cotransfection experiments because both HNF3␣ and - have the capability to recognize the same DNA-binding sequence. Gel-shift experiments suggested that the sequence in the Col18a1 gene, between ⫺2794 bp and ⫺2751 bp, is able to interact simultaneously with members of 2 families of transcription factors, i.e., the liver-enriched transcription factor HNF3 and the ubiquitous factor NF1/CTF. NF1/CTF has been shown to bind to several promoters and to collaborate with tissue-specific and regulated transcription factors. Thus, overlaps between HNF3 and NF1/CTF factors have been described both in the liver-specific vitellogenin gene49 and the eH site of the albumin promoter.32 In the latter, NF1 and HNF3 sites are in close apposition, as we found in the Col18a1 gene, and NF1 can inhibit transcriptional activation by HNF3␣ at an isolated eH site. By contrast, in the albumin enhancer, the 2 factors work cooperatively to enhance transcription. This suggests that increased binding by HNF3 proteins in the enhancer might favor protein-protein interactions
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HEPATOLOGY December 2000
FIG. 7. Analysis of the HNF3/ NF1 binding activity in hepatocellular carcinomas. Nuclear extracts were prepared from 3 normal livers (controls, lanes 1 to 3) and 6 hepatocellular carcinomas (HCC, lanes 4 to 9) and incubated with 32P-labeled oligonucleotides HNF3C18, NF1C18, or HNF3NF1C18. The complexes comigrated with the previously described H1, H2, and X complexes in shift assays with HepG2 cells.
that preclude inhibition of HNF3 by NF1/CTF and that use an inherent transcriptional stimulatory property of NF1/ CTF.50-52 The well-differentiated mhAT3F hepatoma cell line22 is a model system for analyzing the network of liver-enriched transcription factors and their target genes in hepatocytes. Turning off HNF3 activity in this cell line by overproduction of a truncated HNF3 lacking transactivating activity, which competes with endogenous HNF3 proteins for cognate DNAbinding sites, results in a dramatic decrease of liver-specific genes.23 Accordingly, the hepatoma cell line mhATF, stably transfected with a truncated form of HNF3 was found to express collagen ␣1(XVIII) mRNA at a very low level. Together with previous observations, these data favor the concept that Col18a1 gene belongs to the family of liver-specific genes. These findings support our recent studies on the expression of collagen XVIII in both normal and pathological human livers. Indeed, the liver is a main site of collagen XVIII expression in man, and we have shown that hepatocytes in normal livers, and hepatic stellate cells in fibrotic livers, were important sources of this collagen type. Interestingly collagen XVIII expression was high and stable in normal livers and in samples taken from patients with active stage I liver fibrosis and quiescent cirrhosis.14 In addition, the expression of collagen XVIII was increased in well-differentiated hepatocellular carcinomas (Musso et al, manuscript submitted). Accordingly, we found an increase in HNF3/NF1 binding activities in human hepatocellular carcinomas compared with control livers. Interestingly, HNF3/NF1 binding complexes appeared similar to those identified in HepG2. Thus, it is possible that changes in the binding activity of HNF3/NF1 in the promoter 2 of Col18a1 gene in hepatocellular carcinomas result in different transcriptional activity of the gene. In conclusion, our data show that the expression of the collagen XVIII long variant gene is regulated by a combination of liver-enriched and ubiquitous transcription factors. They highlight the specific regulation of this collagen type in the liver and its biological importance, as precursor of endostatin. Acknowledgment: The authors thank for the generous gift of reagents Dr. Schutz, German Cancer Research Center, Heidelberg (Germany); Dr. Suske, Institut fu¨r Molekularbiologie
und Tumorforschung, (Germany); Dr. Costa, University of Illinois at Chicago; and Dr. B. Antoine for providing the mhATF and mhATFS315 cell lines. The authors are indebted to Annie L’Helgoualc’h and Gae¨tan Racine for technical help. REFERENCES 1. Folkman J. Seminars in Medicine of the Beth Israel Hospital, Boston. Clinical applications of research on angiogenesis. N Engl J Med 1995; 333:1757-1763. 2. Bouck N, Stellmach V, Hsu SC. How tumors become angiogenic. Adv Cancer Res 1996;69:135-174. 3. Folkman J. Angiogenesis in cancer, vascular, rheumatoid and other disease. Nature Med 1995;1:27-31. 4. Cle´ment B, Musso O, Lie´tard J, The´ret N. Homeostatic control of angiogenesis: a newly identified function of the liver? Hepatology 1999;29: 621-623. 5. O’Reilly MS, Pirie-Shepherd S, Lane WS, Folkman J. Antiangiogenic activity of the cleaved conformation of the serpin antithrombin. Science 1999;285:1926-1928. 6. Lee TH, Rhim T, Kim SS. Prothrombin kringle-2 domain has a growth inhibitory activity against basic fibroblast growth factor-stimulated capillary endothelial cells. J Biol Chem 1998;273:28805-28812. 7. Colman RW, Jameson BA, Lin Y, Johnson D, Mousa SA. Domain 5 of high molecular weight kininogen (kininostatin) down-regulates endothelial cell proliferation and migration and inhibits angiogenesis. Blood 2000; 95:543-550. 8. O’Reilly MS, Boehm T, Shing Y, Fukai N, Vasios G, Lane WS, Flynn E, et al. Endostatin: an endogenous inhibitor of angiogenesis and tumor growth. Cell 1997;88:277-285. 9. Boehm T, Folkman J, Browder T, O’Reilly MS. Antiangiogenic therapy of experimental cancer does not induce acquired drug resistance. Nature 1997;390:404-407. 10. Sasaki T, Fukai N, Mann K, Gohring W, Olsen BR, Timpl R. Structure, function and tissue forms of the C-terminal globular domain of collagen XVIII containing the angiogenesis inhibitor endostatin. EMBO J 1998;17: 4249-4256. 11. Wen W, Moses MA, Wiederschain D, Arbiser JL, Folkman J. The generation of endostatin is mediated by elastase. Cancer Res 1999;59:60526056. 12. Felbor U, Dreier L, Bryant RA, Ploegh HL, Olsen BR, Mothes W. Secreted cathepsin L generates endostatin from collagen XVIII. EMBO J 2000;19: 1187-1194. 13. Saarela J, Rehn M, Oikarinen A, Autio-Harmainen H, Pihlajaniemi T. The short and long forms of type XVIII collagen show clear tissue specificities in their expression and location in basement membrane zones in humans. Am J Pathol 1998;153:611-626. 14. Musso O, Rehn M, Saarela J, The´ret N, Lie´tard J, Hintikkae E, Lotrian D, et al. Collagen XVIII is localized in sinusoids and basement membrane zones and expressed by hepatocytes and activated stellate cells in fibrotic human liver. Hepatology 1998;28:98-107.
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HEPATOLOGY Vol. 32, No. 6, 2000 15. Saarela J, Rehn M, Oikarinen A, Autio-Harmainen H, Pihlajaniemi T. The short and long forms of type XVIII collagen show clear tissue specificities in their expression and location in basement membrane zones in humans. Am J Pathol 1998;153:611-626. 16. Oh SP, Warman ML, Seldin MF, Cheng SD, Knoll JH, Timmons S, Olsen BR. Cloning of cDNA and genomic DNA encoding human type XVIII collagen and localization of the ␣1(XVIII) collagen gene to mouse chromosome 10 and human chromosome 21. Genomics 1994;19:494-499. 17. Rehn M, Hintikka E, Pihlajaniemi T. Characterization of the mouse gene for the ␣1 chain of type XVIII collagen (Col18␣1) reveals that the three variant N-terminal polypeptide forms are transcribed from two widely separated promoters. Genomics 1996;32:436-446. 18. Muragaki Y, Timmons S, Griffith CM, Oh SP, Fadel B, Quertermous T, Olsen BR. Mouse Col18␣1 is expressed in a tissue-specific manner as three alternative variants and is localized in basement membrane zones. Proc Natl Acad Sci U S A 1995;92:8763-8767. 19. Rehn M, Pihlajaniemi T. Identification of three N-terminal ends of type XVIII collagen chains and tissue-specific differences in the expression of the corresponding transcripts. J Biol Chem 1995;270:4705-4711. 20. Saarela J, Ylikarppa R, Rehn M, Purmonen S, Pihlajaniemi T. Complete primary structure of two variant forms of human type XVIII collagen and tissue-specific differences in the expression of the corresponding transcripts. Matrix Biol 1998;16:319-28. 21. Nordeen SK. Luciferase reporter gene vectors for analysis of promoters and enhancers. Biotechniques 1988;6:454-458. 22. Levrat F, Vallet V, Berbar T, Miquerol L, Khan A, Antoine B. Influence of the content in transcription factors on the phenotype of mouse hepatocyte-like cell lines. Exp Cell Res 1993;209:307-316. 23. Vallet V, Antoine B, Chafey P, Vandemalle A, Khan A. Overproduction of a truncated hepatocyte nuclear factor 3 protein inhibits expression of liver-specific genes in hepatoma cells. Mol Cell Biol 1995;15:5453-5460. 24. Gorman CM, Moffat LF, Howard BH. Recombinant genomes which express chloramphenicol acetyltransferase in mammalian cells. Mol Cell Biol 1982;2:1044-1051. 25. Cereghini S, Blumenfeld M, Yaniv M. A liver-specific factor essential for albumin transcription differs between differentiated and dedifferentiated rat hepatoma cells. Genes Dev 1988;2:957-974. 26. Zaret KS, Liu JK, Dispersio CM. Sites-directed mutagenesis of albumin transcriptional enhancer with the polymerase chain reaction. Proc Natl Acad Sci U S A 1990;87:5469-5473. 27. Costa RH, Grayson DR, Darnell JE. Multiple hepatocyte-enriched nuclear factors function in the regulation of transthyretin and ␣1-antitrypsin genes. Mol Cell Biol 1989;9:1415-1425. 28. Chomczynski P, Saccni N. Single-step method of RNA isolation by guanidinium-thiocyanate-phenol chloroform extraction. Anal Biochem 1987;162:156-169. 29. Lie´tard J, Musso O, Theret N, L’Helgoualc’h A, Campion JP, Yamada Y, Cle´ment B. SP1-mediated transactivation of LAMC1 promoter and coordinated expression of laminin ␥1 and SP1 in human hepatocellular carcinomas. Am J Path 1997;151:1663-1672. 30. Chan YL, Gutell R, Noller HF, Wool IG. The nucleotide sequence of a rat ribosomal ribonucleic acid gene and a proposal for the secondary structure of 18S ribosomal ribonucleic acid. J Biol Chem 1984;259:224-230. 31. Gale´ra P, Park RW, Ducy P, Mattei MG, Karsenty G. c-Krox binds to several sites in the promoter of both mouse type I collagen genes. Structure/function study and developmental expression analysis. J Biol Chem 1996;271:21331-21339. 32. Jackson DA, Rowader KE, Stevens K, Jiang C, Milos P, Zaret KS. Modulation of liver-specific transcription by interactions between Hepatocyte Nuclear Factor 3 and Nuclear Factor 1 binding DNA in close apposition. Mol Cell Biol 1993;13:2401-2410. 33. Karsenty G, Golumbek P, de Crombrugghe B. Point mutations and small substitution mutations in three different upstream elements inhibit the
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activity of the mouse alpha 2(I) collagen promoter. J Biol Chem 1988; 263:13909-13915. Karsenty G, de Crombrugghe B. Two different negative and one positive regulatory factors interact with a short promoter segment of the alpha 1 (I) collagen gene. J Biol Chem 1990;265:9934-9942. Nehls MC, Rippe RA, Veloz L, Brenner DA. Transcription factors nuclear factor I and Sp1 interact with the murine collagen alpha 1 (I) promoter. Mol Cell Biol 1991;11:4065-4073. Tamaki T, Ohnishi K, Hartl C, Le Roy E, Trojanowska M. Characterization of a GC-rich region containing Sp1 binding site(s) as a constitutive responsive element of the ␣2(I) collagen gene in human fibroblasts. J Biol Chem 1995;270:4299-4304. Fischer G, Schmidt C, Opitz J, Cully Z, Ku¨hn K, Po¨schl E. Identification of a novel sequence element in the common promoter region of human collagen type IV genes, involved in the regulation of divergent transcription. Biochem J 1993;292:687-695. Heikkila¨ P, Soininen R, Tryggvason K. Directional regulatory activity of cis-acting elements in the bidirectional ␣1(IV) and ␣2(IV) collagen gene promoter. J Biol Chem 1993;268:24677-24682. Levavasseur F, Lie´tard J, Ogawa K, The´ret N, Burbelo PD, Yamada Y, Guillouzo A, et al. Expression of laminin ␥1 in cultured hepatocytes involves repeated CTC and GC elements in the LAMC1 promoter. Biochem J 1996;313:745-752. Gale´ra P, Musso M, Ducy P, Karsenty G. c-Krox, a transcriptional regulator of type I collagen gene expression, is preferentially expressed in skin. Proc Natl Acad Sci U S A 1994;91:9372-9376. Cereghini S. Liver-enriched transcription factors and hepatocyte differentiation. FASEB J 1996;10:267-282. Hromas R, Costa R. The hepatocyte nuclear factor-3/forkhead transcription regulatory family in development, inflammation, and neoplasia. Crit Rev Oncol Hematol 1995;20:129-140. Nagy P, Bisgaard HC, Thorgeirsson SS. Expression of hepatic transcription factors during liver development and oval cell differentiation. J Cell Biol 1994;126:223-233. Lai E, Prezioso VR, Smith E, Litvin O, Costa RH, Darnell JE. HNF3A, a hepatocyte-enriched transcription factor of novel structure is regulated transcriptionally. Genes Dev 1990;4:1427-1436. Lai E, Prezioso VR, Tao W, Chen WS, Darnell JE. Hepatocyte nuclear factor 3␣ belongs to a gene family in mammals that is homologous to the Drosophila homeotic gene fork head. Genes Dev 1991;5:416-427. Lai E, Clark KL, Burley SK, Darnell JE. Hepatocyte nuclear factor 3/fork head or “winged helix” proteins: a family of transcription factors of diverse biologic function. Proc Natl Acad Sci U S A 1993;90:10421-10423. Liu JK, DiPersio CM, Zaret KS. Extracellular signals that regulate liver transcription factors during hepatic differentiation in vitro. Mol Cell Biol 1991;11:773-784. Pani L, Qian X, Clevidence D, Costa R. The restricted promoter activity of the liver transcription factor hepatocyte nuclear factor 3 involves a cell-specific factor and positive autoactivation. Mol Cell Biol 1992;12: 552-562. Cardinaux JR, Chapel S, Wahli W. Complex organization of CTF/NF1, C/EBP, and HNF3 binding sites within the promoter of the liver-specific vitellogenin gene. J Biol Chem 1994;269:32947-32956. Jones KA, Kadonaga JT, Rosenfeld PJ, Kelly TJ, Tjian R. A cellular DNAbinding protein that activates eukaryotic transcription and DNA replication. Cell 1987;48:79-89. Bru¨ggemeier U, Rogge L, Winnacker EL, Beato M. Nuclear factor I acts as a transcription factor on the MMTV promoter but competes with steroid hormone receptors for DNA binding. EMBO J 1990;9:2233-2239. Graves RA, Tontonoz P, Ross SR, Spiegelman BM. Identification of a potent adipocyte-specific enhancer: involvement of an NF-1-like factor. Genes Dev 1991;5:428-437.
The endostatin precursor collagen XVIII is expressed at high levels in human livers, the main source being hepatocytes. We have studied the regulatory elements in the promoter 2 of the Col18a1 gene that directs the transcription of the NC1-517 variant of collagen ␣1(XVIII), which is the main form expressed in the liver. The 5ⴕ-flanking region of Col18a1 gene was cloned, and a series of 5ⴕ-deletions from ⴚ3286 bp to ⴙ285 bp were linked to the luciferase reporter gene. Transfection experiments in HepG2 cells allowed to identify a silencer-like element containing putative HNF1 and HNF3 sites and activator elements containing stretches of GC-rich sequences. Another putative HNF3 site in close apposition to a NF1/CTF site was localized upstream of the silencer-like element. Cotransfection experiments showed that the Col18a1 promoter 2 was transactivated by Sp1 and HNF3␣. Gel-shift analyses showed that HNF3, NF1/CTF, and Sp1-like sites specifically recognized nuclear factors. Super-shift experiments indicated that HNF3 was the major form of HNF3 interacting with the HNF3/NF1 site. The well-differentiated hepatoma cell line mhATFS315 transfected with a truncated form of HNF3, which competitively blocks HNF3 transactivating activity, expressed the Col18a1gene at a very low level. Taken together, these data strongly suggest that Col18a1 is a liver-specific gene. Furthermore, gel-shift analyses performed with nuclear factors prepared from well-differentiated hepatocellular carcinomas showed increased HNF3/NF1 binding activity compared with normal livers. Consequently, the precursor of endostatin might be differently expressed according to the
Abbreviations: HNF, hepatocyte nuclear factor; CMV, cytomegalovirus; cDNA, complementary DNA; CAT, chloramphenicol acetyl transferase; TLC, thin-layer chromatography; EDTA, ethylenediaminetetraacetic acid; EGTA, ethyleneglycoltetraacetic acid; TBE, tris/borated/EDTA; mRNA, messenger RNA; TTR, transthyretin. From the 1Detoxication and Tissue Repair Unit, INSERM U-456, Universite´ de Rennes I, France; and 2Collagen Research Unit, Biocenter and Department of Medical Biochemistry, University of Oulu, Oulu, Finland. Received May 18, 2000; accepted September 7, 2000. Supported by INSERM, DRRC Bretagne (CHU Pontchaillou, Rennes), Association pour la Recherche contre le Cancer (France), the Health Sciences Council of the Academy of Finland, the Sigrid Juselius Foundation, and FibroGen Inc. (South San Francisco, CA). Sequence data from this article has been submitted to the GenBank/EMBL Data libraries with accession number No 34607. Drs. Lie´tard and The´ret contributed equally to this work. Address reprint requests to: Nathalie The´ret, Ph.D., INSERM U-456, Universite´ de Rennes I, 2 ave. Le´on Bernard, 35043 Rennes Cedex, France. E-mail: [email protected]; fax: (33) 2-99-33-62-42. Copyright © 2000 by the American Association for the Study of Liver Diseases. 0270-9139/00/3206-0024$3.00/0 doi:10.1053/jhep.2000.20066
differentiated and/or transformed state of hepatocytes. (HEPATOLOGY 2000;32:1377-1385.) Angiogenesis is involved in most physiological and pathological processes and is essential for tumor growth and metastasis.1,2 It depends on a balance between stimulatory factors, e.g., vascular endothelial growth factor and fibroblast growth factors, and inhibitory factors, e.g., thrombospondin-1, SPARC, fibronectin fragments, angiostatin, and endostatin.3 We have recently proposed that the endogenous regulation of angiogenesis is a newly identified liver function,4 as endostatin, angiostatin, the cleaved form of antithrombin,5 the kringle-2 domain of prothrombin,6 and the domain 5 of kininogen7 are cryptic angiogenesis inhibitors within parent plasma proteins involved in the clotting and fibrinolytic cascades and are secreted by hepatocytes. Endostatin8,9 is proteolytically derived from the C-terminal domain of the nonfibrillar collagen XVIII10-12 which is associated with basement membranes13 and is secreted as a plasma protein.10 Collagen XVIII is expressed at high levels by human adult hepatocytes, and its expression is increased early in the onset of liver fibrosis.14,15 Human and mouse collagen ␣1(XVIII) genes are located on chromosome 21q22.3 and 10, respectively.16 The complete organization of the 102 kb mouse Col18a1 gene was recently reported.17 Mouse collagen XVIII transcripts encode polypeptides that differ in size with respect to the 3 variant N-terminal noncollagenous domains that are 301 (NC1-301), 517 (NC1517), or 764 (NC1-764) residues in length.18,19 The 3 variant NC1 domains result from the use of 2 alternative promoters, separated by a distance of 50 kb.17 The upstream promoter, promoter 1, directs the synthesis of the NC1-301 domain. The downstream promoter, promoter 2, directs that of the NC1517 and NC1-764 domains, with the latter 2 variants differing with respect to alternative splicing.17 Human hepatocytes express the NC1-517 variant of collagen ␣1(XVIII),13,14 which is almost exclusively detected in adult and fetal livers at high levels, suggesting that its expression is liver-specific in humans.20 However, neither the 5⬘-flanking regions of the Col18a1 gene nor the factors that govern its striking hepatocyte-specific expression have been characterized so far. In the present study, we have identified potential regulatory elements implicated in the regulation of the transcription of Col18a1 gene in HepG2 cells after cloning the 5⬘-flanking region of promoter 2, which governs the expression of the NC1-517 variant. We show that, as in other genes specifically expressed in the liver, both Sp1 (ubiquitous) and HNF3/NF1 (liver-specific) regulatory elements are involved in the combinatorial control of the expression of collagen XVIII and that
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HNF3/NF1 binding activity is increased in well-differentiated human hepatocellular carcinomas. MATERIALS AND METHODS Sequence Analysis of the 5ⴕ-Flanking Region of the Downstream Promoter From the Mouse Collagen ␣1(XVIII) Gene A 5.3-kb HindIII fragment of the P1 clone P1-212417 was digested with NaeI, and the resulting 3.6-kb HindIII/NaeI fragment covering the promoter 2 sequences of the Col18a1 was subcloned into the vector pSP72 (Promega, Madison, WI). Sequencing from both strands was performed with ABI PRISM 377 Automated Sequencer (Perkin-Elmer, Applied Biosystems, France) and consensus sites for the binding of transcription factors were searched with Mat Inspector Prog. (Transfac database). Plasmid Constructs Several recombinant plasmids containing the 5⬘-flanking sequences of the gene coding for the 2 longest variants of collagen XVIII fused to the structural part of the gene encoding luciferase were constructed. The vector pSP72 has been first modified as follows: pSP72 was linearized with XhoI/EcoRI, a linker containing restriction sites was attached (XhoI-SalI-MluI-HindIII-NaeI-EcoRI), digested with HindIII/NaeI and then the 3.6-kb HindIII/NaeI fragment mentioned earlier was ligated. The plasmid was then digested with MluI and protected with dNTPs, digested with HindIII and the double-stranded deletions were prepared using the “double-stranded nested deletion kit” (Pharmacia) according to the manufacturer. Segment deletions were selected and subcloned into the SalI/SmaI site of pXP2 vector.21 The 3.6-kb fragment in pSP72 and the different deletion constructs used in this study, i.e., del 7 (⫺3286 bp to ⫹285 bp), del 9 (⫺2835 bp to ⫹285 bp), del 10 (⫺1843 bp to ⫹285 bp), del 1.3 (⫺1329 bp to ⫹285 bp), del 2.1 (⫺935 bp to ⫹285 bp), and del 12 (⫺492 bp to ⫹285 bp), were sequenced from both strands using ABI PRISM 377 Automated Sequencer (Perkin-Elmer). The human expression vector for Sp1 under the control of the CMV promoter, pEVR2/CMV/Sp1 and pCMV-HNF3␣ were generous gifts of Dr. Suske (Institut fu¨r Molekularbiologie und Tumorforschung, Germany) and Dr. Costa (University of Illinois at Chicago), respectively. All plasmids used for transfection were purified by cesium chloride centrifugation steps. Cell Culture Human hepatoblastoma (HepG2) cells were routinely maintained in Williams’s medium (Eurobio, France) supplemented with 10% fetal calf serum and 1% streptomycin at 37°C. Only confluent cells were used in the study. The well-differentiated mhAT3F hepatoma cell line was derived from the tumorous liver of a transgenic mouse expressing simian virus 40 early genes under the control of the liverspecific antithrombin III promoter.22 The mhAT3F S315 consisted of stably transfected mhAT3F with a truncated HNF3 protein expression vector resulting in competition with the endogenous HNF3 proteins and subsequently in inhibition of liver-specific genes.23 Both the wild type, mhAT3F, and the transfected mhATFS315 hepatoma cell lines were obtained from Dr. B. Antoine (INSERM U129, Institut Cochin, Paris) and cultured as previously described.23 Liver Samples Liver tissue samples were obtained from patients at the Department of Surgery, Hoˆpital Pontchaillou, Rennes, France, between November 1994 and July 1997. Tumor samples from 6 patients with hepatocellular carcinoma were analyzed. Normal samples (n ⫽ 3) were from liver donors who had been excluded from the transplantation protocol because of extra-abdominal infection unrelated to the cause of death. The absence of significant histological lesions was verified before inclusion of these samples as controls. All specimens were routinely processed for histology, i.e., hematoxylin-eosin-saffran and Sirius red staining. Access to this biopsy material was in
HEPATOLOGY December 2000 TABLE 1. Oligonucleotides Used in This Study Sp1C18 (⫺818 bp) Sp1cons HNF3C18 (⫺2788 bp) HNF3alb HNF3TTr NF1C18 (⫺2778 bp) HNF3NF1C18 (⫺2778 bp)
5⬘-GGGGTCAGGGCGGCTATGTG-3⬘ 5⬘-ATTCGATCGGGGCGGGGCGAGC-3⬘ 5⬘-TCCAGCTGTTTGCAGCTGGA-3⬘ 5⬘-CCGAACGTGTTTGCCTTGGCC-3⬘ 5⬘-GTTGACTAAGTCAATAATCAGAATCAG 5⬘-AGCTGGACTCTGAGCCAAA-3⬘ 5⬘-TCCAGCTGTTTGCAGCTGGACTCTGAGCCAAA-3⬘
NOTE. Binding sites containing sequences from Col18␣1 promoters (Sp1C18, HNF3C18, NF1C18, and HNF3NF1C18) and from albumin (HNF3alb) and transthyretin (HNF3TTr) gene promoters are underlined. Sp1 consensus is commercially available from Santa Cruz Biotechnology.
agreement with French laws and satisfied the requirements of the Ethics Committee of the institution. Transient Transfections and Luciferase Assays HepG2 cells were transfected with plasmid DNA by electroporation using a Gene Pulser apparatus (Eurogentec, Seraing, Belgium). Luciferase plasmids (30 g) were cotransfected with 10 g of pRSVCAT plasmid to normalize for transfection efficiencies. For cotransfection experiments, 30 g of luciferase plasmids were cotransfected with either 10 g of pEVR2/CMV/Sp1 plasmid, 10 g of pCMV/ HNF3␣ plasmid or 10 g of the control expression vectors (without the respective cDNA). Cells were harvested 48 hours after transfection, and luciferase activity was determined from cell extracts using the luciferase assay system (Promega, Madison, WI). CAT assays were carried out by the method of Gorman et al.24 Radioactive spots on TLC plates were cut out and quantitated by scintillation counting. Nuclear Protein Extracts and Gel Shift Assays Nuclear extracts from HepG2 cells and from liver samples were prepared, and gel shift assays were performed as described by Cereghini et al.25 Binding reactions were carried out in a 15 L volume containing 1 mmol/L sodium phosphate, pH 7.5, 0.1 mmol/L ethylenediaminetetraacetic acid (EDTA), 0.5 mmol/L ethyleneglycoltetraacetic acid (EGTA), 0.5 mmol/L dithiothreitol, 10% (vol/vol) glycerol, 2 g poly (dI-dC), 1 mmol/L MgCl2, 10 mmol/L spermidine, and 20,000 cpm of P-labeled 5⬘-end double-stranded oligonucleotide. Oligonucleotides from the Col18a1 gene promoter 2, and from both HNF3 albumin26 and HNF3 transthyretin27 gene promoters are presented in Table 1. The Sp1 consensus oligonucleotide was purchased from Santa Cruz Biotechnology and was termed Sp1cons. Five micrograms of nuclear proteins were added to the reaction mixture and incubated at room temperature for 20 minutes. The DNA-protein complexes were resolved on 6% acrylamide gels in 0.5 ⫻ TBE (45 mmol/L Tris-borate, 1.25 mmol/L EDTA) at 25 mA. Then, gels were fixed, dried, and subjected to autoradiography. For supershift experiments, 5 g of nuclear proteins and radiolabeled oligonucleotides were incubated at room temperature for 20 minutes in 10 mmol/L Tris (pH 7.5) buffer with 50 mmol/L NaCl, 1 mmol/L dithiothreitol, 1 mmol/L EDTA, 5% glycerol and 1 g poly (dI-dC). One microliter of specific anti HNF3␣, HNF3, or HNF3␥ antibody (kindly provided by Dr. G. Schutz, German Cancer Research Center, Heidelberg, Germany) and 1 or 5 L of specific antiNF1/CTF antibodies (kindly provided by Dr. M. Raymondjean, INSERM U129, Institut Cochin, Paris) were then added and incubated at room temperature for 45 minutes. Northern and Dot Blot Analyses Total RNA from cells were prepared by using the guanidium thiocyanate/phenol-chloroforme method.28 Northern and dot blots were
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FIG. 1. Sequence of the 5⬘-flanking region of the promoter 2 from the mouse Col18a1 gene coding the polypeptide forms NC1-517 and NC1-764. The nucleotides are numbered from the extreme upstream major transcription initiation site. Horizontal bars indicate the locations of consensus sequences for binding sites of certain transcription factors. The CTC-rich sequences are in italics.
performed as previously described.29 cDNA Collagen XVIII probe was a 458 bp cDNA, which recognized the long variant of the mouse ␣1(XVIII) chains.19 A 25-mer oligoprobe corresponding to 18S ribosomal RNA was used30 for standardization of the signals.
latter being in close apposition with a NF1/CTF site at ⫺2778 bp.
RESULTS
Potential regulatory elements responsible for governing transcription of the Col18a1 gene were identified in HepG2 cells. A series of 5⬘ deletions from ⫺3286 bp to ⫹285 bp was constructed from the mouse promoter and linked to the luciferase reporter gene. Transfection experiments were carried out in 3 independent experiments (Fig. 2). The del 7 plasmid gave a transcriptional activity that was arbitrarily chosen as 100%. The del 9 plasmid showed a slightly lower activity. Truncation of the upstream region from ⫺2835 bp to ⫺1329 bp resulted in increased luciferase activities after transfection of del 10 and del 1.3, respectively. Truncation of the putative Sp1-binding site localized at ⫺1068 bp, and transfection of del 2.1 construct resulted in a weaker luciferase activity compared with the del 1.3 plasmid. Transfection of the shortest construct, del 12 from ⫺492 bp to ⫹285 bp gave a marked decrease in luciferase activity. This suggests that a silencerlike element(s) is present between ⫺2835 bp and ⫺1329 bp,
Sequence Analysis of the 5ⴕ-Flanking Region of the Downstream Promoter From the Mouse Collagen ␣1(XVIII) Gene
The sequence of 3.4 kb upstream of the transcription start site of the promoter 2 was determined (Fig. 1). The gene lacked a TATA box upstream of the transcription start site, but contained 2 CCAAT boxes at ⫺65 bp and ⫺148 bp, with the latter one in reverse orientation. The gene also contained several CTC-rich sequences, including a stretch between ⫺2058 bp and ⫺2170 bp and 3 boxes localized at ⫺1026 bp, ⫺1855 bp, and ⫺3049 bp, respectively. Potential binding sites for transcription factors were identified including several Sp1 binding sites, 1 AP-1 binding site, and 2 potential binding sites for c-Krox factor.31 In addition, 1 HNF1 potential binding site was found at ⫺1918 bp, and 2 HNF3 binding sites were localized at ⫺1746 bp and ⫺2788 bp, respectively, the
Deletion Analysis of the Col18a1 Promoter
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FIG. 2. Transfection analysis of the Col18a1 promoter 2 in HepG2 cell line. A series of deletion constructs and their corresponding luciferase activities are represented on the left. Relative luciferase activities are given as percentages of del 7 plasmid activity. The different deletion plasmids were cotransfected with the expression vectors coding HNF3␣ and Sp1. The transactivation of the different fragments was evaluated by measuring the ratio of the relative luciferase activity in presence of the expression vector versus that obtained in presence of the control vector (right panel).
and a transcriptional activator element between ⫺1329 bp and ⫺492 bp, which contains several GC-rich elements and an AP-1 site. Cotransfections With Sp1 and HNF3␣ Expression Vectors
To investigate whether Sp1 and HNF3␣ have a potential role in the regulation of the Col18a1 gene, the different luciferase constructs were cotransfected with corresponding expression vectors. Results for each deletion construct are expressed as a ratio of the relative luciferase activity obtained with the expression vector to that obtained with the control (Fig. 2). Cotransfection with the Sp1 expression vector markedly increased the activity of all the constructs containing GC-rich elements. The 2 longest constructs containing the upstream HNF3 site, i.e., del 7 and particularly del 9, were
FIG. 3. (A) Gel shift assays. Nuclear extracts were prepared from HepG2 cells and incubated with specific oligonucleotides. (A, left panel) 32P-labeled oligonucleotide Sp1C18 (lanes 2 to 4): Incubation steps were performed without (-) or with 100fold excess of Sp1C18 (lane 3) and Sp1cons (lane 4) unlabeled oligonucleotides. Lane 1: control without nuclear extracts. Arrows on the left indicate specific complexes. NS, non-specific complex. (A, right panel) 32P-labeled oligonucleotides SP1consensus (lanes 1 to 3). Incubation steps were performed with 100 (lane 2) or 50 fold-excess (lane 3) of Sp1C18 oligonucleotides. (B) Northern blot analyses of endogenous collagen ␣1(XVIII) mRNA expression in HepG2 cells transfected with either pEVR2-CMV-Sp1 (lane 2) or pEVR2-CMV (lane 3). Controls were electroporated cells in the absence of plasmid (lane 1). The ratio collagen XVIII/18S were evaluated by densitometry scanning.
induced by HNF3␣, whereas the activity of other plasmids was not changed. These results suggest that the Col18a1 gene is transactivated with Sp1 and that HNF3␣ can be implicated in the regulation of the Col18a1 gene through the upstream HNF3 site located at ⫺2788 bp. Sp1 Binding Activity in HepG2 Cells
The interactions of endogenous Sp1 factor(s) with the Col18a1 promoter was investigated by gel-shift assays with HepG2 nuclear factors. Oligonucleotide-termed Sp1C18 (Table 1) correspond to sequence in the promoter region containing putative Sp1-binding site localized at ⫺818 bp. Two complexes (arrows in Fig. 3A, left panel, lane 2) were resolved by gel-shift assay with 32P-labeled Sp1C18 probe. These complexes were competed by a 100-fold molar excess of unlabeled
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competitor Sp1C18 (lane 3) or Sp1consensus (lane 4). Complexes of similar sizes were found with SP1cons oligonucleotides (Fig. 3A, right panel, lane 1) and were competed by a 100-fold molar excess of unlabeled competitor Sp1C18 (lane2), the 2 Sp1-specific complexes being still detectable in the presence of 20-fold molar excess of competitor (Fig. 3A, right panel, lane 3). These results suggest that the GC-rich sequence at ⫺818 bp interacts with Sp1-related factor(s). To assess whether Sp1 modulate collagen ␣1(XVIII) mRNA expression, HepG2 cells were transfected with a Sp1 expression vector (Fig. 3B). Control was electroporated cells in the absence of plasmid, and assays were carried out with either pEVR2-CMV-Sp1 or pEVR2-CMV construct. The steady-state collagen ␣1(XVIII) mRNA levels were 2.3-fold and 3.6-fold increased when cells were transfected with Sp1 expression vector (lane 2) compared with control (lane 1) and Sp1-less expression vector (lane 3), respectively. This suggests that Sp1 acts as a positive regulatory element for the transcription of Col18a1 gene. HNF3 and NF1/CTF Binding Activity
The recognition sequence for the NF1/CTF family of transcription factors, TGGAN7CCA is located 3 bp downstream of the HNF3 putative site localized at ⫺2788 bp. Because it has been shown in other systems that interactions between HNF3 and NF1 in close apposition occurs in liver-specific genes,32 gel-shift experiments were performed with different oligonucleotides (Table 1). The first corresponds to the HNF3 site at ⫺2788 bp in the Col18a1 gene (oligonucleotide HNF3C18), the second corresponds to the 2 putative HNF3 and NF1/CTF sites in close apposition (oligonucleotide HNF3NF1C18). The third corresponds to the NF1/CTF site (oligonucleotide NF1C18). HNF3C18. Two major complexes, termed H1 and H2, were detected by gel-shift assay with 32P-labeled HNF3C18 oligonucleotide (Fig. 4A, lanes 1 and 11). H1 complexes were competed by a 60-fold excess of unlabeled HNF3C18 oligonucleotides (lanes 2 and 14) whereas competition with H2 complexes required higher concentration of oligonucleotides (lanes 3 and 4). Binding of HNF3C18 was only partially competed by oligonucleotides corresponding to the binding sites in the mouse transthyretin (HNF3TTR) and albumin (HNF3alb) genes (lanes 5 to 10). The complex H1 was completely competed by an excess of unlabeled HNF3NF1C18 (Fig 4A, lane 12), and slightly competed by an excess of unlabeled NF1C18 (lane 13), the second complex H2 being partially competed by HNF3NF1C18 and NF1C18 oligonucleotides (lanes 12 and 13, respectively). NF1C18. Two complexes were detected by gel-shift assay with 32P-labeled NF1C18 oligonucleotide (Fig. 4B, lanes 1 and 4). These complexes had the same shift than complexes H1 and H2, H1 being totally and H2 partially competed by an excess of both cold HNF3NF1C18 (lane 5) and NF1 (lane 6) oligonucleotides. An excess of cold HNF3C18 oligonucleotide did not show competition (lane 7). HNF3NF1C18. Three complexes were detected by gel-shift assay with 32P-labeled HNF3NF1C18 oligonucleotide (Fig. 4B, lanes 3 and 8), including H1 and H2 and an additional complex, X. The complex X was completely competed by an excess of unlabeled HNF3NF1C18 (Fig. 4B, lane 9), NF1C18 (lane 10) and HNF3C18 oligonucleotides (lane 11). Complex H1 was totally competed by an excess of cold HNF3NF1C18 (lane 9), being only partially reduced by an excess of cold
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NF1C18 (lane 10) and not competed by an excess of cold HNF3C18 oligonucleotides (lane 11). The signal intensity of complex H2 was reduced by HNF3NF1C18 and NF1C18 oligonucleotides (lane 9 and 10, respectively) but not affected by HNF3C18 oligonucleotides (lane 11). Taken together, the data indicate that several nuclear factors are involved in the formation of the 2 complexes H1 and H2, which interact with HNF3- and NF1-like regulatory elements between ⫺2794 bp and ⫺2751 bp. Complex X probably corresponds to a supra-arrangement of nuclear factors, which cooperate for interacting with the HNF3/NF1 site. Importantly, the data suggest that both HNF3- and NF1-binding sites overlaps, as previously shown in other liver-specific genes. The affinity of complex interactions with HNF3/NF1like motif appears modulated by NF1-related factors. Involvement of HNF3 in the Regulation of Col18a1 Expression
To further characterize the complexes H1, H2, and X, nuclear extracts from HepG2 were incubated with radiolabeled HNF3C18 or HNF3NF1C18 oligonucleotides in the presence of either specific anti-HNF3␣, HNF3, HNF3␥, or NF1 antibodies (Fig. 5). The lower intensity of complexes H2 in supershift assay conditions compared with those in shift assays may be related to the differences in stringency between the 2 reaction mixtures, which greatly affect stability of complexes H2. When anti-HNF3 antibodies were added to the reaction mixture with radiolabeled HNF3C18 (Fig. 5A), a specific decrease in the intensity of the H1 complex was observed, the intensity of complex H2 being enhanced (Fig. 5A, lane 3). No significant change was found with either HNF3␣ or HNF3␥ antibodies (Fig. 5A, lanes 2 and 4). A similar pattern was observed when HNF3TTR and HNF3alb oligonucleotides were used as probes (Fig. 5A). With HNF3NF1C18 and NF1C18 oligonucleotides, similar shifts of H1 and H2 complexes were observed in the presence of HNF3 antibodies (Fig. 5B, lanes 3 and 9). In addition, the complex X was strongly reduced. Addition of NF1 antibodies prevented the formation of complex X with HNF3NF1C18 oligonucleotides (Fig. 5B, lanes 5 and 6); a slight decrease in the intensity of complex H1 was found with NF1C18 oligonucleotides (Fig. 5B, lane 12), being more obvious with HNF3C18 oligonucleotides (Fig. 5B, lane 18). Despite a low intensity of H2 complexes in supershift assay conditions, NF1 antibodies appeared to affect the formation of H2 complexes (Fig. 5B, lane 18). The expression of collagen ␣1 (XVIII) was investigated in the mouse hepatoma cell line mhATF. This cell line was stably transfected with a truncated form of HNF3, which competed with endogenous HNF3 for binding DNA sites, thus resulting in a decrease in hepatospecific gene expression.23 As shown in Fig. 6, the steady-state collagen ␣1(XVIII) mRNA was significantly lower in transfected versus nontransfected cells, similarly to albumin mRNA levels. Taken together, the data indicate that HNF3 is the major form of HNF3 involved in the formation of the protein complexes that regulate Col18a1 promoter 2 in HepG2 cells and that this regulatory factor is necessary for a stable expression of Col18a1 gene. In addition, HNF3␣ is also able to recognize the regulatory elements as shown by the induction of del 7 and del 9 transcription by HNF3␣ expression vector. Thus, Col18a1 gene appears to be regulated by nuclear factors that modulate liver-specific gene expression.
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Changes in HNF3/NF1 Binding Activity in Hepatocellular Carcinomas
The significance of HNF3/NF1 binding activity was further studied in human liver biopsies by gel shift assays. Nuclear extracts from 3 control livers and 6 hepatocellular carcinomas were incubated with radiolabeled oligonucleotides (Fig. 7). Three complexes comigrating with those observed in HepG2 cells nuclear extracts were found: H1, H2, and X, the intensity of the latter being very low in control liver and weaker in tumor samples compared with HepG2 cells. A striking difference between tumor and control livers was an increase in binding activities in hepatocellular carcinoma samples, thus suggesting the involvement of these complexes in regulating collagen ␣1 (XVIII) expression in cancer liver. DISCUSSION
We have analyzed the molecular mechanisms that govern the expression of the ␣1 chain of collagen XVIII, by cloning the promoter 2 of Col18a1 gene that encodes the NC1-517 domain, i.e., the major collagen XVIII variant in the liver. We found that the 5⬘-flanking region of the downstream promoter 2 of Col18a1 gene contains 2 CCAAT boxes and several putative Sp1-binding sites, which are also found in the regulatory regions of both COL1A1 and COL1A2 that encode collagen ␣1(I) and ␣2(I) transcripts, respectively33-36 and in collagen IV genes.37,38 Different putative Sp1-binding sites were identified, and one of them located at ⫺818 bp is able to bind an Sp1-related factor(s). An Sp1 expression vector was able to transactivate the different constructs that contain GC-rich boxes and to act as a positive regulator on basal collagen ␣1 (XVIII) mRNA expression in the human hepatoma cell line, HepG2. In addition, several CTC boxes, another element found in the regulatory regions of various collagen genes, including COL1A1,34,35 COL1A2,33 COL4A1,37,38 and LAMC1 that encodes the laminin ␥1 chain39 were also present. Two potential binding sites for c-Krox with the consensus sequence GGGAGGG31 were also identified. It has been shown that c-Krox can bind to several sites in the promoter of both mouse collagen I genes,31,40 but whether the 2 sites in the Col18a1 gene are involved in its regulation needs further investigation. Besides these ubiquitous sites found in the regulatory regions of various collagen and housekeeping genes, potential binding sites for the liver-enriched transcription factors HNF1 and HNF3 were identified in the promoter 2 of Col18a1. Genes that are preferentially expressed in the liver, e.g., albumin, ␣-antitrypsin, aldolase B41 are under the combinatorial control of several families of liver-enriched transcription factors, including the leucine zipper C/EBP 4™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™ Fig. 4. Analysis of the 2 putative HNF3 and NF1/CTF binding sites in close apposition in the Col18a1 promoter 2. Nuclear extracts were prepared from HepG2 cells and incubated with 32P-labeled oligonucleotides. (A) Competition of HNF3C18 binding site by 60, 300, and 1,000 molar excess of unlabeled oligonucleotides. HNF3C18 lanes 2, 3, and 4; HNF3TTR lanes 5, 6, and 7; and HNF3alb, lanes 8, 9, and 10, respectively; by an excess of cold competitor HNF3NF1C18 (lane 12), NF1C18 (lane 13), HNF3C18 (lane 14) oligonucleotides. (B) Comparison of complex formation by gel shift assays with NF1C18 (lane 1), HNF3C18 (lanes 2), HNF3NF1C18 (lanes 3). Complexes H1, H2, and X are indicated on the left. Competition of NF1C18 and HNF3NF1C18 binding sites by an excess of cold competitors HNF3NF1C18 (lanes 5 and 9), NF1C18 (lanes 6 and 10), and HNF3C18 (lanes 7 and 11) oligonucleotides.
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FIG. 5. Gel mobility supershift assays with HNF3 and NF1 antibodies. Nuclear extracts were prepared from HepG2 cells and incubated with 32P-labeled oligonucleotides. (A) HNF3C18 (lanes 1 to 4), HNF3TTRC18 (lanes 5 to 8), and HNF3alb (lanes 9 to 12). Incubation steps were performed without (lanes 1, 5, and 9) or with HNF3␣ (lanes 2, 6, and 10), HNF3 (lanes 3, 7, and 11), HNF3␥ (lanes 4, 8, and 12) antibodies. (B) HNF3C18 (lanes 1 to 6), NF1C18 (lanes 7 to 12), and HNFNF1C18 (lanes 13 to 18). Incubation steps were performed without (lanes 1, 7, and 13) or with HNF3␣ (lanes 2, 8, and 14), HNF3 (lanes 3, 9, and 15), HNF3␥ (lanes 4, 10, and 16), NF1 (1 L) (lanes 5, 11, and 17), NF1 (5 L) (lanes 6, 12, and 18) antibodies. Specific complexes H1, H2, and X are indicated on the left. The lower staining intensity of complex H2 compared with that in Fig. 4 is related to the differences in buffer composition.
(CCAAT/enhancer binding protein), HNF3, HNF1, and HNF4 families.41,42 These families are characterized by structurally related, DNA-binding domains that control the formation of the hepatic endoderm during development and the phenotype of adult hepatocytes.43 The architecture of these promoter regions consists of clusters of liver-specific and ubiquitous transcription factors within short regions, which allow the regulation of transcription through the synergy of both classes of transactivators. We found that HNF3 in combination with NF1/CTF are important regulator elements of the murine Col18a1 promoter 2 activity. Importantly, a putative HNF3-NF1/CTF site is also present in the promoter 2 of the human COL18A1 gene, at about the same distance upstream of the transcription start site mouse (Elamaa H., Lie´tard J. and Pihlajaniemi T., unpublished data, 1999). The HNF3 family is composed of 3 isoforms, namely HNF3␣, HNF3, and HNF3␥, which are encoded by 3 distinct genes that are believed to participate in the liver-specific expression
FIG. 6. Dot blot analyses of endogenous expression of collagen XVIII in the mhATF (䊐) and in the deficient HNF3 mhATFS315 (■) cell lines. Albumin mRNA was used as hepatospecific gene control. Values from densitometric scanning were expressed as mean ⫾ SD of Collagen ␣1(XVIII)/18S and albumin/18S ratios (n ⫽ 5). Student’s t test was used to test the significance of the difference between means: **P ⬍ .01; ***P ⬍ .001.
of a number of genes.27,44,45 This transcription factor family is characterized by a highly conserved DNA-binding domain.46 HNF3␣, , and ␥ recognize 2 apparently unrelated DNA sequence motives, 1 is defined by the high-affinity site of the transthyretin (TTR) promoter,27 the other being the TGT3 motif.32,47,48 In the present study, we show that cotransfection with the HNF3␣ expression vector resulted in an induced activity of the 2 constructs containing the upstream HNF3 site. In apparent contradiction, a silencer-like region was identified between ⫺2835 bp and ⫺1329 bp, as indicated by transfection experiments of deleted plasmids. These observations suggest that the silencer element(s) is likely located downstream of the HNF3 site at ⫺2788 bp. In support of this, the HNF3 expression vector had no effect on the del 10 construct, which contains the second HNF3 site, located at ⫺1746 bp. Thus, the upstream HNF3 site, i.e., at ⫺2788 bp might positively regulate the activity of the promoter. Interestingly, super-shift experiments indicated that HNF3 was the major HNF3 polypeptide in HepG2 to bind the HNF3 sequence in this region. This does not contradict data from cotransfection experiments because both HNF3␣ and - have the capability to recognize the same DNA-binding sequence. Gel-shift experiments suggested that the sequence in the Col18a1 gene, between ⫺2794 bp and ⫺2751 bp, is able to interact simultaneously with members of 2 families of transcription factors, i.e., the liver-enriched transcription factor HNF3 and the ubiquitous factor NF1/CTF. NF1/CTF has been shown to bind to several promoters and to collaborate with tissue-specific and regulated transcription factors. Thus, overlaps between HNF3 and NF1/CTF factors have been described both in the liver-specific vitellogenin gene49 and the eH site of the albumin promoter.32 In the latter, NF1 and HNF3 sites are in close apposition, as we found in the Col18a1 gene, and NF1 can inhibit transcriptional activation by HNF3␣ at an isolated eH site. By contrast, in the albumin enhancer, the 2 factors work cooperatively to enhance transcription. This suggests that increased binding by HNF3 proteins in the enhancer might favor protein-protein interactions
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FIG. 7. Analysis of the HNF3/ NF1 binding activity in hepatocellular carcinomas. Nuclear extracts were prepared from 3 normal livers (controls, lanes 1 to 3) and 6 hepatocellular carcinomas (HCC, lanes 4 to 9) and incubated with 32P-labeled oligonucleotides HNF3C18, NF1C18, or HNF3NF1C18. The complexes comigrated with the previously described H1, H2, and X complexes in shift assays with HepG2 cells.
that preclude inhibition of HNF3 by NF1/CTF and that use an inherent transcriptional stimulatory property of NF1/ CTF.50-52 The well-differentiated mhAT3F hepatoma cell line22 is a model system for analyzing the network of liver-enriched transcription factors and their target genes in hepatocytes. Turning off HNF3 activity in this cell line by overproduction of a truncated HNF3 lacking transactivating activity, which competes with endogenous HNF3 proteins for cognate DNAbinding sites, results in a dramatic decrease of liver-specific genes.23 Accordingly, the hepatoma cell line mhATF, stably transfected with a truncated form of HNF3 was found to express collagen ␣1(XVIII) mRNA at a very low level. Together with previous observations, these data favor the concept that Col18a1 gene belongs to the family of liver-specific genes. These findings support our recent studies on the expression of collagen XVIII in both normal and pathological human livers. Indeed, the liver is a main site of collagen XVIII expression in man, and we have shown that hepatocytes in normal livers, and hepatic stellate cells in fibrotic livers, were important sources of this collagen type. Interestingly collagen XVIII expression was high and stable in normal livers and in samples taken from patients with active stage I liver fibrosis and quiescent cirrhosis.14 In addition, the expression of collagen XVIII was increased in well-differentiated hepatocellular carcinomas (Musso et al, manuscript submitted). Accordingly, we found an increase in HNF3/NF1 binding activities in human hepatocellular carcinomas compared with control livers. Interestingly, HNF3/NF1 binding complexes appeared similar to those identified in HepG2. Thus, it is possible that changes in the binding activity of HNF3/NF1 in the promoter 2 of Col18a1 gene in hepatocellular carcinomas result in different transcriptional activity of the gene. In conclusion, our data show that the expression of the collagen XVIII long variant gene is regulated by a combination of liver-enriched and ubiquitous transcription factors. They highlight the specific regulation of this collagen type in the liver and its biological importance, as precursor of endostatin. Acknowledgment: The authors thank for the generous gift of reagents Dr. Schutz, German Cancer Research Center, Heidelberg (Germany); Dr. Suske, Institut fu¨r Molekularbiologie
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